The heterogeneity in drug sensitivity among tumor cell subpopulations is a major obstacle in cancer chemotherapy and remains poorly understood. The goal of this proposal is to develop a spheroid model that can be used to study tumor heterogeneity. Spheroids possess many characteristics of an in situ tumor, yet have the advantage of an in vitro system in that they can be grown and assayed under rigidly controlled culture conditions. We will grow spheroids from defined mixtures of 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitive and resistant 9L rat brain tumor cells. These spheroids will be evaluated in terms of cell survival, growth delay and sister chromatid exchange (SCE) induction. Using the cell survival and growth delay assays, BCNU effectiveness will be described as a function of the percent of resistant cells in the spheroid. Interactions affecting the growth rate of the individuals cell subpopulations will be investigated using the growth delay assay. The SCE assay will be used to quantitate the proportion and BCNU sensitivity of each cell type in the spheroid. Therefore, based on SCE induction, we will be able to detect changes in the proportions and drug sensitivities of the cell types during spheroid growth. In addition, the SCE assay will be used in combination with sequential disaggregation to determine the location of the cell subpopulations within the spheroid.